Hepatitis-c virus type 4, 5 and 6

ABSTRACT

The present invention relates to a polynucleic acid composition comprising or consisting of at least one polynucleic acid containing 8 or more contiguous nucleotides corresponding to a nucleotide sequence from the region spanning positions 417 to 957 of the Core/E1 region of HCV type 3; and/or the region spanning positions 4664 to 4730 of the NS3 region of HCV type 3; and/or the region spanning positions 4892 to 5292 of the NS3/4 region of HCV type 3; and/or the region spanning positions 8 023 to 8 235 of the NS5 region of the BR36 subgroup of HCV type 3 a ; and/or the coding region of HCV type 4 a  starting at nucleotide 379 in the core region; and/or the coding region of HCV type 4; and/or the coding region of HCV type 5, with said nucleotide numbering being with respect to the numbering of HCV nucleic acids as shown in Table 1, and with said polynucleic acids containing at least one nucleotide difference with known HCV type 1, and/or HCV type 2 genomes in the above-indicated regions, or the complement thereof.

TECHNICAL FIELD

The present invention relates to newly elucidated sequences of hepatitisC virus type 4 (HCV-4), and type 5 (HCV-5), and to a newly discoveredtype 6 (HCV-6). In particular, it relates to the etiologic agent ofhepatitis C virus type 4, 5 and 6, and to polynucleotides andimmunoreactive polypeptides which are useful in immunoassays for thedetection of HCV-4, HCV-5 and HCV-6 in biological samples; and also tothe use of antigenic HCV-4, HCV-5 and HCV-6 specific polypeptides invaccines.

BACKGROUND OF THE INVENTION

Acute viral hepatitis is a disease which may result in chronic liverdamage. It is clinically diagnosed by a well-defined set of patientsymptoms, including jaundice, hepatic tenderness, and an increase in theserum levels of alanine aminotransferase and aspartate aminotransferase.Serologic immunoassays are generally performed to diagnose the specifictype of viral causative agent. Historically, patients presenting withsymptoms of hepatitis and not otherwise infected by hepatitis A,hepatitis B, Epstein-Barr or cytomegalovirus were clinically diagnosedas having non-A, non-B hepatitis (NANBH) by default.

For many years, the agent of non-A, non-B hepatitis remained elusive. Ithas now been established that many cases of NANBH are caused by adistinct virus termed hepatitis C virus (HCV). European PatentApplication EP-A-0318216 discloses cDNA sequences derived from onestrain of HCV, polynucleotide probes and polypeptides for use inimmunoassays. Further information on that strain is provided in EuropeanApplication EP-A-0388232.

The HCV genome encodes a large polyprotein precursor, which containsstructural and non-structural regions. The single protein is apparentlycleaved into a variety of proteins after production. Most of thestructural and non-structural proteins have now been identified from invitro RNA translation and expression as recombinant proteins. The C andE regions encode for nucleocapsid structural proteins and for envelopestructural proteins, respectively. At least five additional regionsfollow, which encode for non-structural (NS) protein of undefinedfunction. The organisation is believed to be as follows (A. Alberti,Journal of Hepatology, 1991; 12; 279 to 282)5′                                           3′ NCR: C : E1 : E2 : NS1 :NS2 : NS3 : NS4 : NS5Certain immunoreactive proteins have been described as recombinantproteins, for example C22 (in the core region), C33 (in NS3 region),5-1-1 and C100 (both in the NS4 region), and NS5 (NS5 region). Currentdiagnosis of hepatitis C is often based on methods which detectantibodies against the product of the C-100 clone. This clone wasproduced by ligation of overlapping clones to produce a larger viralantigen (C100) corresponding to part of the NS3-NS4 genomic region. C100was then fused with the human superoxide dismutase (SOD) gene, expressedin use as a large recombinant fusion protein (C100-3) and used on solidphase to develop radio-labelled (RIA) and enzyme-linked immunosorbentassays (ELISA).

Polynucleotides alleged to be useful for screening for HCV are disclosedin European Patent Specification EP-A-0398748. European PatentSpecification EP-A-0414475 purports to disclose the propagation of HCVin culture cells and the production of antigens for use in diagnostics.European Patent Specification EP-A-0445423 discloses what is stated tobe an improved immunoassay for detecting HCV antibodies.

Blood banks in the United Kingdom carry out routine testing of blooddonors for antibodies to components of HCV. A first generation assayinvolved the detection of HCV antibodies to C100-3 polypeptides. TheC100-3 antibody recognises a composite polyprotein antigen withinnon-structural regions of the virus and is a consistent marker of HCVinfection. However, in acute infections this antibody is unreliablebecause of the delay (typically 22 weeks) in seroconversion afterexposure. Furthermore, the C100-3 antibody test lacks specificity forthe hepatitis C virus.

Second generation antibody tests employ recombinant antigens and/orsynthetic linear peptides representing structural antigens from thehighly conserved core region of the virus as well as non-structuralantigens. However, it is found that some second-generation ELISA testscan yield false-positive reactions. The recombinant immunoblot assay(RIBA-2) incorporating four antigens from the HCV genome, purports toprovide a method for identifying genuine-anti-HCV reactivity. However,the result can be “indeterminate”. The present workers have reported(The Lancet, 338; Oct.19, 1991) varying reactivity of HCV-positive blooddonors to 5-1-1, C100, C33 and C22 antigens, and compared these with theresults of the direct detection of HCV RNA present in the blood samplesusing polymerase chain reaction (PCR) to amplify HCV polynucleotides.However, the work demonstrates that the unambiguous diagnosis of HCVinfections is not yet possible.

Recently there has been discovered further types of HCV that differconsiderably in sequence and these have been called HCV-2, 3 and 4. Ourpatent application WO93/10239 (published on 27th May, 1993) describescertain antigenic sequences of HCV-2, 3 and 4. The sequences disclosedfor HCV-4 are in the 5′ NCR and core regions only. The former does notcode for any protein which might be used in an immunoassay for HCVwhilst the core region tends to be conserved.

SUMMARY OF THE INVENTION

The present invention includes the discovery of a previously unknowntype 6 variant of HCV, by a comparison of sequences amplified bypolymerase chain reaction (PCR) in certain regions of the HCV genome andconfirmed by phylogenetic analysis. The invention has identifiedpolynucleotide sequences and polypeptides which are HCV-4, HCV-5 andHCV-6 specific. These may be used to diagnose HCV-4, HCV-5 and HCV-6infection and should thus be included in any definitive test for HCVinfection.

One aspect of the present invention provides a polynucleotide having anucleotide sequence unique to hepatitis virus type 4, 5 or 6.

The sequences are unique to the HCV type concerned in the sense that thesequence is not shared by any other HCV type, and can thus be used touniquely detect that HCV-type. Sequence variability between HCV 4, 5 and6 has been found particularly in the NS4, NS5 and core regions and it istherefore from these regions in particular that type-specificpolynucleotides and peptides may be obtained. The term type-specificindicates that a sequence unique to that HCV type is involved. Moreover,within each HCV type a number of sub-types may exist having minorsequence variations.

The invention includes NS5 polynucleotide sequences unique to hepatitisC virus types 4 and 6 (HCV-4 and HCV-6); and NS4 sequences unique toHCV-4, HCV-5 and HCV-6 respectively. The sequences may be RNA or DNAsequences, including cDNA sequences. If necessary the DNA sequences maybe amplified by polymerase chain reaction. DNA sequences can be used asa hybridisation probe. The sequences may be recombinant (i.e. expressedin transformed cells) or synthetic and may be comprised within longersequences if necessary. Equally, deletions, insertions or substitutionsmay also be tolerated if the polynucleotide may still function as aspecific probe. Polynucleotide sequences which code for antigenicproteins are also particularly useful.

Another aspect of the invention provides a peptide having an amino acidsequence unique to hepatitis virus type 4, 5 or 6.

The invention includes antigenic HCV-4 or HCV-6 specific polypeptidefrom the NS5 region, or antigenic HCV-4, HCV-5 or HCV-6 specificpolypeptide from the NS4 region; or polypeptides including theseantigens. A plurality of copies of the peptide may be bound to amultiple antigen peptide core.

The peptide may be labelled to facilitate detection, and may for examplebe labelled antigenic HCV-4 or HCV-6 specific polypeptide from the NS5region, or labelled antigenic HCV-4, HCV-5 or HCV-6 specific polypeptidefrom the NS4 region; (or mixtures thereof) for use in an immunoassay todetect the corresponding antibodies.

It should be understood that the polypeptides will not necessarilycomprise the entire NS4 or NS5 region, but that characteristic partsthereof (usually characteristic epitopes) unique to a particular type ofHCV may also be employed.

A further aspect of the invention provides antibodies to the peptides,especially to HCV-4 or HCV-6 NS5 antigens, or to HCV-4, HCV-5 or HCV-6NS4 antigens, particularly monoclonal antibodies for use in therapy anddiagnosis. Thus labelled antibodies may be used for in vivo diagnosis.Antibodies carrying cytotoxic agents may be used to attack HCV-4, HCV-5or HCV-6 infected cells.

A further aspect of the invention provides a vaccine comprisingimmunogenic peptide, especially HCV-4 or HCV-6 NS5 polypeptide, orimmunogenic HCV-4, HCV-5 or HCV-6 NS4 polypeptide.

A further aspect of the invention provides a method of in vitro HCVtyping which comprises carrying out endonuclease digestion of anHCV-containing sample to provide restriction fragments, the restrictionpattern being characteristic of HCV-4, HCV-5 or HCV-6.

Finally, the present invention also encompasses assay kits includingpolypeptides which contain at least one epitope of HCV-4, HCV-5 or HCV-6antigen (or antibodies thereto), as well as necessary preparativereagents, washing reagents, detection reagents and signal producingreagents.

The HCV-4, HCV-5 or HCV-6 specific polynucleotide sequences may be usedfor identification of the HCV virus itself (usually amplified by PCR) byhybridisation techniques.

Oligonucleotides corresponding to variable regions, e.g. in the NS5 orNS4 region, could be used for type-specific PCR. Outer sense and innersense primers may be used in combination with the two conservedanti-sense primers for a specific detection method for HCV types 4, 5and 6.

The present invention also provides expression vectors containing theDNA sequences as herein defined, which vectors being capable, in anappropriate host, of expressing the DNA sequence to produce the peptidesas defined herein.

The expression vector normally contains control elements of DNA thateffect expression of the DNA sequence in an appropriate host. Theseelements may vary according to the host but usually include a promoter,ribosome binding site, translational start and stop sites, and atranscriptional termination site. Examples of such vectors includeplasmids and viruses. Expression vectors of the present inventionencompass both extrachromosomal vectors and vectors that are integratedinto the host cell's chromosome. For use in E. coli, the expressionvector may contain the DNA sequence of the present invention optionallyas a fusion linked to either the 5′- or 3′-end of the DNA sequenceencoding, for example, B-galactosidase or the 3′-end of the DNA sequenceencoding, for example, the trp E gene. For use in the insect baculovirus(AcNPV) system, the DNA sequence is optionally fused to the polyhedrincoding sequence.

The present invention also provides a host cell transformed withexpression vectors as herein defined.

Examples of host cells of use with the present invention includeprokaryotic and eukaryotic cells, such as bacterial, yeast, mammalianand insect cells. Particular examples of such cells are E.coli,S.cerevisiae, P.pastoris, Chinese hamster ovary and mouse cells, andSpodoptera frugiperda and Tricoplusia ni. The choice of host cell maydepend on a number of factors but, if post-translational modification ofthe HCV viral peptide is important, then an eukaryotic host would bepreferred.

The present invention also provides a process for preparing a peptide asdefined herein which comprises isolating the DNA sequence, as hereindefined, from the HCV genome, or synthesising DNA sequence encoding thepeptides as defined herein, or generating a DNA sequence encoding thepeptide, inserting the DNA sequence into an expression vector such thatit is capable, in an appropriate host, of being expressed, transforminghost cells with the expression vector, culturing the transformed hostcells, and isolating the peptide.

The DNA sequence encoding the peptide may be synthesised using standardprocedures (Gait, Oligonucleotide Synthesis: A Practical Approach, 1984,Oxford, IRL Press).

The desired DNA sequence obtained as described above may be insertedinto an expression vector using known and standard techniques. Theexpression vector is normally cut using restriction enzymes and the DNAsequence inserted using blunt-end or staggered-end ligation. The cut isusually made at a restriction site in a convenient position in theexpression vector such that, once inserted, the DNA sequences are underthe control of the functional elements of DNA that effect itsexpression.

Transformation of an host cell may be carried out using standardtechniques. Some phenotypic marker is usually employed to distinguishbetween the transformants that have successfully taken up the expressionvector and those that have not. Culturing of the transformed host celland isolation of the peptide as required may also be carried out usingstandard techniques.

The peptides of the present invention may thus be prepared byrecombinant DNA technology, or may be synthesized, for example by usingan automatic synthesizer.

The term “peptide” (and “polypeptide”) is used herein to includeepitopic peptides having the minimum number of amino acid residues forantigenicity, through oligopeptides, up to proteins. The peptide may bea recombinant peptide expressed from a transformed cell, or could be asynthetic peptide produced by chemical synthesis.

Antibody specific to a peptide of the present invention can be raisedusing the peptide. The antibody may be used in quality control testingof batches of the peptides; purification of a peptide or viral lysate;epitope mapping; when labelled, as a conjugate in a competitive typeassay, for antibody detection; and in antigen detection assays.

Polyclonal antibody against a peptide of the present invention may beobtained by injecting a peptide, optionally coupled to a carrier topromote an immune response, into a mammalian host, such as a mouse, rat,sheep or rabbit, and recovering the antibody thus produced. The peptideis generally administered in the form of an injectable formulation inwhich the peptide is admixed with a physiologically acceptable diluent.Adjuvants, such as Freund's complete adjuvant (FCA) or Freund'sincomplete adjuvant (FIA), may be included in the formulation. Theformulation is normally injected into the host over a suitable period oftime, plasma samples being taken at appropriate intervals for assay foranti-HCV viral antibody. When an appropriate level of activity isobtained, the host is bled. Antibody is then extracted and purified fromthe blood plasma using standard procedures, for example, by protein A orion-exchange chromatography.

Monoclonal antibody against a peptide of the present invention may beobtained by fusing cells of an immortalising cell line with cells whichproduce antibody against the viral of topographically related peptide,and culturing the fused immortalised cell line. Typically, a non-humanmammalian host, such as a mouse or rat, is inoculated with the peptide.After sufficient time has elapsed for the host to mount an antibodyresponse, antibody producing cells, such as the splenocytes, areremoved. Cells of an immortalising cell line, such as a mouse or ratmyeloma cell line, are fused with the antibody producing cells and theresulting fusions screened to identify a cell line, such as a hybridoma,that secretes the desired monoclonal antibody. The fused cell line maybe cultured and the monoclonal antibody purified from the culture mediain a similar manner to the purification of polyclonal antibody.

Diagnostic assays based upon the present invention may be used todetermine the presence of absence of HCV infection, and the HCV typeinvolved. They may also be used to monitor treatment of such infection,for example in interferon therapy.

In an assay for the diagnosis of viral infection, there are basicallythree distinct approaches that can be adopted involving the detection ofviral nucleic acid, viral antigen or viral antibody respectively. Viralnucleic acid is generally regarded as the best indicator of the presenceof the virus itself and would identify materials likely to beinfectious. However, the detection of nucleic acid is not usually asstraightforward as the detection of antigens or antibodies since thelevel of target can be very low. Viral antigen is used as a marker forthe presence of virus and as an indicator of infectivity. Depending uponthe virus, the amount of antigen present in a sample can be very low anddifficult to detect. Antibody detection is relatively straightforwardbecause, in effect, the host immune system is amplifying the response toan infection by producing large amounts of circulating antibody. Thenature of the antibody response can often be clinically useful, forexample IgM rather than IgG class antibodies are indicative of a recentinfection, or the response to a particular viral antigen may beassociated with clearance of the virus. Thus the exact approach adoptedfor the diagnosis of a viral infection depends upon the particularcircumstances and the information sought. In the case of HCV, adiagnostic assay may embody any one of these three approaches.

In any assay for the diagnosis of HCV involving detection of viralnucleic acid, the method may comprise hybridising viral RNA present in atest sample, or cDNA synthesised from such viral RNA, with a DNAsequence corresponding to the nucleotide sequences of the presentinvention or encoding a peptide of the invention, and screening theresulting nucleic acid hybrids to identify any HCV viral nucleic acid.The application of this method is usually restricted to a test sample ofan appropriate tissue, such as a liver biopsy, in which the viral RNA islikely to be present at a high level. The DNA sequence corresponding toa nucleotide sequence of the present invention or encoding a peptide ofthe invention may take the form of an oligonucleotide or a cDNA sequenceoptionally contained within a plasmid. Screening of the nucleic acidhybrids is preferably carried out by using a labelled DNA sequence.Preferably the peptide of the present invention is part of anoligonucleotide wherein the label is situated at a sufficient distancefrom the peptide so that binding of the peptide to the viral nucleicacid is not interfered with by virtue of the label being too close tothe binding site. One or more additional rounds of screening of one kindor another may be carried out to characterise further the hybrids andthus identify any HCV viral nucleic acid. The steps of hybridisation andscreening are carried out in accordance with procedures known in theart.

A further method for the detection of viral nucleic acid involvesamplification of a viral DNA using polymerase chain reaction (PCR). Theprimers chosen may be specific to the HCV type s quence of interest, sothat amplification occurs only with that particular HCV type. Also thesize and number of amplified copy sequences may be characteristic ofparticular HCV types, or they may have characteristic restrictionpatterns with chosen endonucleases.

In an assay for the diagnosis of HCV involving detection of viralantigen or antibody, the method may comprise contacting a test samplewith a peptide of the present invention or a polyclonal or monoclonalantibody against the peptide and determining whether there is anyantigen-antibody binding contained within the test sample. For thispurpose, a test kit may be provided comprising a peptide, as definedherein, or a polyclonal or monoclonal antibody thereto and means fordetermining whether there is any binding with antibody or antigenrespectively contained in the test sample to produce an immune complex.The test sample may be taken from any of appropriate tissue orphysiological fluid, such as blood (serum or plasma), saliva, urine,cerebrospinal fluid, sweat, tears or tissue exudate. If a physiologicalfluid is obtained, it may optionally be concentrated for any viralantigen or antibody present.

A variety of assay formats may be employed. The peptide can be used tocapture selectively antibody against HCV from solution, to labelselectively the antibody already captured, or both to capture and labelthe antibody. In addition, the peptide may be used in a variety ofhomogeneous assay formats in which the antibody reactive with thepeptide is detected in solution with no separation of phases.

The types of assay in which the peptide is used to capture antibody fromsolution involve immobilization of the peptide on to a solid surface.This surface should be capable of being washed in some way. Examples ofsuitable surfaces include polymers of various types (moulded intomicrotitre wells; beads; dipsticks of various types; aspiration tips;electrodes; and optical devices). particles (for example latex;stabilized red blood cells; bacterial or fungal cells; spores; gold orother metallic or metal-containing sols; and proteinaceous colloids)with the usual size of the particle being from 0.02 to 5 microns,membranes (for example of nitrocellulose; paper; cellulose acetate; andhigh porosity/high surface area membranes of an organic or inorganicmaterial).

The attachment of the peptide to the surface can be by passive adsoptionfrom a solution of optimum composition which may include surfactants,solvents, salts and/or chaotropes; or by active chemical bonding. Activebonding may be through a variety of reactive or activatible functionalgroups which may be exposed on the surface (for example condensingagents; active acid esters, halides and anhydrides; amino, hydroxyl, orcarboxyl groups; sulphydryl groups; carbonyl groups; diazo groups; orunsatruated groups). Optionally, the active bonding may be through aprotein (itself attached to the surface passively or through activebonding), such as albumin or casein, to which the viral peptide may bechemically bonded by any of a variety of methods. The use of a proteinin this way may confer advantages because of isoelectric point, charge,hydrophilicity or other physico-chemical property. The viral peptide mayalso be attached to the surface (usually but not necessarily a membrane)following electrophorectic separation of a reaction mixture, such asimmunoprecipitation.

In the present invention it is preferred to provide blocking peptideswhich block any cross-reactivity and leave only those HCV antibodies inthe sample which will react solely with the type of antigen present inthat particular test location. For example, a test location intended todetect HCV-6 will be blocked by a blocking mixture comprising HCV-1 to 5peptides which will react with all antibodies having reactivity to HCVtypes 1 to 5 and leave antibodies having only type 6 reactivity.

After contacting the surface bearing the peptide with a test sample (inthe presence of a blocking mixture if required), allowing time forreaction, and, where necessary, removing the excess of the sample by anyof a variety of means, (such as washing, centrifugation, filtration,magnetism or capilliary action) the captured antibody is detected by anymeans which will give a detectable signal. For example, this may beachieved by use of a labelled molecule or particle as described abovewhich will react with the captured antibody (for example protein A orprotein G and the like; anti-species or anti-immunoglobulin-sub-type;rheumatoid factor; or antibody to the peptide, used in a competitive orblocking fashion), or any molecule containing an epitope contained inthe peptide. In the present invention, it is preferred to add ananti-human IgG conjugated to horseradish peroxidase and then to detectthe bound enzyme by reaction with a substrate to generate a colour.

The detectable signal may be produced by any means known in the art suchas optical or radioactive or physico-chemical and may be provideddirectly by labelling the molecule or particle with, for example, a dye,radiolabel, fluorescent, luminescent, chemiluminescent, electroactivespecies, magnetically resonant species or fluorophore, or indirectly bylabelling the molecule or particle with an enzyme itself capable ofgiving rise to a measurable change of any sort. Alternatively thedetectable signal may be obtained using, for example, agglutination, orthrough a diffraction or birefrigent effect if the surface is in theform of particles.

Assays in which a peptide itself is used to label an already capturedantibody require some form of labelling of the peptide which will allowit to be detected. The labelling may be direct by chemically orpassively attaching for example a radiolabel, magnetic resonant species,particle of enzyme label to the peptide; or indirect by attaching anyform of label to a molecule which will itself react with the peptide.The chemistry of bonding a label to the peptide can be directly througha moiety already present in the peptide, such as an amino group, orthrough an intermediate moiety, such as a maleimide group. Capture ofthe antibody may be on any of the surfaces already mentioned in anyreagent including passive or activated adsorption which will result inspecific antibody or immune complexes being bound. In particular,capture of the antibody could be by anti-species oranti-immunoglobulin-sub-type, by rheumatoid factor, proteins A, G andthe like, or by any molecule containing an epitope contained in thepeptide.

The labelled peptide may be used in a competitive binding fashion inwhich its binding to any specific molecule on any of the surfacesexemplified above is blocked by antigen in the sample. Alternatively, itmay be used in a non-competitive fashion in which antigen in the sampleis bound specifically or non-specifically to any of the surfaces aboveand is also bound to a specific bi- or poly-valent molecule (e.g. anantibody) with the remaining valencies being used to capture thelabelled peptide.

Often in homogeneous assays the peptide and an antibody are separatelylabelled so that, when the antibody reacts with the recombinant peptidein free solution, the two labels interact to allow, for example,non-radiative transfer of energy captured by one label to the otherlabel with appropriate detection of the excited second label or quenchedfirst label (e.g. by fluorimetry, magnetic resonance or enzymemeasurement). Addition of either viral peptide or antibody in a sampleresults in restriction of the interaction of the labelled pair and thusin a different level of signal in the detector.

A further possible assay format for detecting HCV antibody is the directsandwich enzyme immunoassay (EIA) format. An antigenic peptide is coatedonto microtitre wells. A test sample and a peptide to which an enzyme iscoupled are added simultaneously. Any HCV antibody present in the testsample binds both to the peptide coating the well and to theenzyme-coupled peptide. Typically, the same peptide are used on bothsides of the sandwich. After washing, bound enzyme is detected using aspecific substrate involving a colour change.

It is also possible to use IgG/IgM antibody capture ELISA wherein anantihuman IgG and/or IgM antibody is coated onto a solid substrate. Whena test sample is added, IgG and/or IgM present in the sample will thenbind to the antihuman antibody. The bound IgG and/or IgM represents thetotal population of those antibodies. A peptide of the present inventionwill bind only to those IgG and/or IgM antibodies that were produced inresponse to the antigenic determinant(s) present in the peptide i.e. tothose antibodies produced as a result of infection with the type of HCVfrom which the peptide was derived. For detection of thepeptide/antibody complex the peptide may itself have been labelleddirectly or, after interaction with the captures antibodies, the peptidemad be reacted with a labelled molecule that binds to the peptide.

It can thus be seen that the peptides of the present invention may beused for the detection of HCV infection in many formats, namely as freepeptides, in assays including classic ELISA, competition ELISA, membraneb bound EIA and immunoprecipitation. Peptide conjugates may be used inamplified assays and IgG/IgM antibody capture ELISA.

An assay of the present invention may be used, for example, forscreening donated blood or for clinical purposes, for example, in thedetection, typing and monitoring of HCV infections. For screeningpurposes, the preferred assay formats are those that can be automated,in particular, the microtitre plate format and the bead format. Forclinical purposes, in addition to such formats, those suitable forsmaller-scale or for single use, for example, latex assays, may also beused. For confirmatory assays in screening procedures, antigens may bepresented on a strip suitable for use in Western or other immunoblottingtests.

As indicated above, assays used currently to detect the presence ofanti-HCV antibodies in test samples, particularly in screening donatedblood, utilise antigenic peptides obtained from HIV type 1 only and suchantigens do not reliable detect other HCV genotypes. Accordingly, it isclearly desirable to supplement testing for HIV-1 with testing for allother genotypes, for example, types 2, 3, 4, 5 and 6 and also anyfurther genotypes that may be discovered.

In particular, the invention allows blood donor screening byconventional assays (using HCV type 1 encoded antigens) to besupplemented with a second test that contains oligopeptidescorresponding to antigenic regions found for example in the NS5 sequenceof HCV-4 or HCV-6, or the NS4 sequence of HCV-4, HCV-5 or HCV-6.

To test for a spectrum of genotypes, there may be provided a series ofassay means each comprising one or more antigenic peptides from onegenotype of HCV, for example, a series of wells in a microtitre plate,or an equivalent series using the bead format. Such an assay format maybe used to determine the type of HCV present in a sample. Alternatively,or in addition, an assay means may comprise antigenic peptides from morethan one type, for example, a microwell or bead may be coated withpeptides from more than one type.

Oligopeptides corresponding to the antigenic regions of HCV-4, HCV-5 orHCV-6 may also be used separately to distinguish individuals infectedwith these different HCV types. Such an assay could be in the format ofan indirect enzyme immunoassay (EIA) that used sets of wells or beadscoated with oligopeptides of the antigenic regions for HCV types 4, 5and 6. Minor degrees of cross-reactivity, should they exist, can beabsorbed out by dilution of the test serum in a diluent that containedblocking amounts of soluble heterologous-type oligopeptides, to ensurethat only antibody with type-specific antibody reactivity bound to thesolid phase.

It may be advantageous to use more than one HCV antigen for testing, inparticular, a combination comprising at least one antigenic peptidederived from the structural region of the genome and at least oneantigenic peptide derived from the non-structural region, especially acombination of a core antigen and at least one antigen selected from theNS3, NS4 and NS5 regions. The wells or beads may be coated with theantigens individually. It may be advantageous, however, to fuse two ormore antigenic peptides as a single polypeptide, preferably as arecombinant fusion polypeptide. Advantages of such an approach are thatthe individual antigens can be combined in a fixed, predetermined ratio(usually equimolar) and that only a single polypeptide needs to beproduced, purified and characterised. One or more such fusionpolypeptides may be used in an assay, if desired in addition to one ormore unfused peptides. It will be appreciated that there are manypossible combinations of antigens in a fusion polypeptide, for example,a fusion polypeptide may comprise a desired range of antigens from onetype only, or may comprise antigens from more than one type.

To obtain a polypeptide comprising multiple peptide antigens by anexpression technique, one approach is to fuse the individual codingsequences into a single open reading frame. The fusion should, ofcourse, be carried out in such a manner that the antigenic activity ofeach component peptide is not significantly compromised by its positionrelative to another peptide. Particular regard should of course be hadfor the nature of the sequences at the actual junction between thepeptides. The resulting coding sequence can be expressed, for example,as described above in relation to recombinant peptides in general. Themethods by which such a fusion polypeptide can be obtained are known inthe art, and the production of a recombinant fusion polypeptidecomprising multiple antigens of a strain of HCV type 1 is described inGB-A-2 239 245. Peptide conjugates may be used in amplified assays andIgG/IgM antibody capture ELISA.

The peptide of the present invention may be incorporated into a vaccineformulation for inducing immunity to HCV in man. The vaccine may includeantigens of HCV types 1 to 6. For this purpose the peptide may bepresented in association with a pharmaceutically acceptable carrier.

For use in a vaccine formulation, the peptide may optionally bepresented as part of an hepatitis B core fusion particle, as describedin Clarke et al (Nature, 1987, 330, 381-384), or a polylysine basedpolymer, as described in Tam (PNAS, 1988, 85, 5409-5413).

Alternatively, the peptide may optionally be attached to a particulatestructure, such as lipsomes or ISCOMS.

Pharmaceutically, acceptable carriers for the vaccine include liquidmedia suitable for use as vehicles to introduce the peptide into apatient. An example of such liquid media is saline solution. The peptidemay be dissolved or suspended as a solid in the carrier.

The vaccine formulation may also contain an adjuvant for stimulating theimmune response and thereby enhancing the effect of the vaccine.Examples of adjuvants include aluminium hydroxide and aluminiumphosphate.

The vaccine formulation may contain a final concentration of peptide inthe range from 0.01 to 5 mg/ml, preferably from 0.03 to 2 mg/ml. Thevaccine formulation may be incorporated into a sterile container, whichis then sealed and stored at a low temperature, for example 4° C., ormay be freeze-dried.

In order to induce immunity in man to HCV, one or more doses of thevaccine formulation may be administered. Each dose may be 0.1 to 2 ml,preferably 0.2 to 1 ml. A method for inducing immunity to HCV in man,comprises the administration of an effective amount of a vaccineformulation, as hereinbefore defined.

The present invention also provides the use of a peptide as hereindefined in the preparation of a vaccine for use in the induction ofimmunity to HCV in man.

Vaccines of the present invention may be administered by any convenientmethod for the administration of vaccines including oral and parenteral(e.g. intravenous, subcutaneous or intramuscular) injection. Thetreatment may consist of a single dose of vaccine or a plurality ofdoses over a period of time.

DESCRIPTION OF THE DRAWINGS

Examples of the invention will now be described by way of example only.

FIG. 1 is a comparison of inferred amino acid sequences of part of theNS5 region of HCV types 4 and 6 (in comparison to type 1a) fromExample 1. The number of sequences compared are shown in the secondcolumn. Single amino acid codes are used. The position and frequency ofvariability within an HCV type is indicated by a subscript;

FIG. 2 gives corresponding DNA nucleotide sequences in the NS5 region ofHCV-4 (three sequences) and of HCV-6 (one sequence);

FIG. 3 is a phylogenetic analysis of NS5 sequences from 67 isolates ofHCV, showing major HCV types (numbered 1-6) and subtypes (designateda,b,c) and demonstrating that HCV-4 and HCV-6 are distinct types. Thesequence distance is proportional to the spacing on the tree accordingto the indicated scale.

FIG. 4 shows DNA primer sequences as used in Example 2 for the PCRamplification of two regions of the NS4 region of HCV-4, HCV-5 andHCV-6; and

FIG. 5 shows DNA and amino acid sequences for two partial regions of theNS4 region of HCV-4, HCV-5 and HCV-6 deduced from the nucleotidesequences elucidated in Example 2; for comparison the correspondingregions of HCV-1, 2 and 3 are given, HCV-3 regions 1 and 2 correspondingto amino acids 1691-1708 and 1710-1728 respectively of FIG. 9 b ofWO93/10239 (see also Simmonds et al., 1993, J. Clin. Microb. 31:1493).

EXAMPLE 1 (HCV-4 and HCV-6; NS5 Region Sequences)

Samples. Plasma from a total of 16 HCV-infected blood donors inScotland, Egypt and Hong Kong and from a patient with chronic hepatitisin Lebabon were used for analysis of NS5 sequences of types 4 and 6.

Nucleotide sequence analysis. To obtain sequences in the NS5 region,viral RNA was reverse transcribed and amplified by polymerase chainreaction (PCR) in a single reaction with previously published primersthought to be highly conserved amongst different variants of HCV(Enomoto et al. 1990). For some sequences, a second PCR was carried outwith primers 554 and 555 (Chan et al., 1992b) in combination with twonew primers, 122 (sense orientation; 5′ CTC AAC CGT CAC TGA GAG AGA CAT3′) and 123 (anti-sense; 5′ GCT CTC AGG TTC CGC TCG TCC TCC 3′). ProductDNA was phosphorylated, purified and cloned into the SmaI site of pUC19(Yanisch-Perron et al., 1985) following the procedures described inSimmonds & Chan, 1993. Alternatively, amplified DNA was purified anddirectly sequenced as described in Simmonds et al., 1990 and Cha et al.,1992. These methods allowed comparison of a 222 bp fragment of DNAhomologous to positions 7975 to 8196 in the prototype virus (numbered asin Choo et al., 1991). The results are shown in FIGS. 1 and 2 Nucleotidesequence comparisons. Nucleotide sequences were aligned using theCLUSTAL V program (Higgins et al. 1992) as implemented in the GDEsequence analysis package. Distances between pairs of sequences wereestimated using the DNADIST program of the PHYLIP package (version 3,4)kindly provided by Dr. J. Felsenstein (Felsenstein, 1991), using a modelwhich allow different rates of transition and tranversion and differentfrequences of the four nucleotides (Felsenstein, 1991). Phylogenetictrees were constructed using the neighbour-joining algorithm on theprevious sets of pairwise distances (Saitou et al. 1987) using thePHYLIP program, NEIGHBOR. The phylogenetic tree shown in FIG. 3 isunrooted. Equivalent phylogenetic relationships were also found in amaximum likelihood analysis (PHYLIP program DNAMI; data not shown), and200 bootstrap replicates of neighbour-joining trees (PHYLIP programsSEQBOOT and CONSENSE).

EXAMPLE 2 (HCV-4, -5 and -6; NS4 Region Sequences)

Attempts have been made to isolate DNA sequences from the NS4 region ofHepatitis C virus (HCV) types 4, 5 and 6 using PCR amplification. Thedecision was made to use primers which contained restriction sites,thereby allowing the cloning of the PCR products via cohesive endcloning. New primers were also designed from relatively conservedregions of the HCV NS4 gene. The cloning strategy involved severalparticular steps, as follows.

-   (i) Klenow repair. The termini of the amplified DNA were repaired    with Klenow DNA polymerase to ensurethat the ends which contained    the restriction sites were complete.-   (ii) Kinasing of termini. The PCR product termini were    phosphorylated using T4 polynucleotide kinase. This allowed    self-ligation of the products in a concatemerization step.-   (iii) Concatemerization of the PCR products. The DNA fragments were    ligated together to form long concatemeric arrays. This step    internalized the restriction sites encoded in the ends of the    primers which greatly increased the efficiency of the cleavage step.-   (iv) Restriction digestion. The PCR products were digested overnight    with the required restriction enzyme to expose the cohesive ends.-   (v) Ligation to the plasmid vector.

General procedures and reagents are described in Maniatis et al.“Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory,Cold Spring Harbor: N.Y.

a) PCR Amplification of NS4 Sequences

After cDNA synthesis using primer 007, three rounds of PCR amplificationwere carried out. Types 4 and 5 were amplified by one round using 007,and 435, followed by two rounds using 5351 and 5943 (which both encodeBamHI restriction sites). Type 6 sequences were amplified by one roundusing 007 plus 253, 281 and 221, followed by two rounds using 5351 and5943. The products of a minimum of 5 third round reactions were pooledfor the cloning steps. The efficiency of the final ligation into thevector appeared to be dependant on a high concentration of the amplifiedDNA. The primer sequences are listed in FIG. 4.

b) Cloning

The PCR products were isolated by excision from a conventional agarosegel (0.5× tris acetate EDTA (TAE)). The DNA was reclaimed from theagarose by centrifugation through glass wool. The eluates were pooled inorder to increase the total amount of DNA. The DNA was retrieved fromthe TAE eluate by ethanol precipitation. (The pelleted DNA containedsome chemically inert debris from the agarose but this did not interferewith the following steps prior to ligation into the vector. Magic prepcolumns (Promega) could be used, but ethanol precipitation is simpler,cheaper and more efficient if dealing with a large volume of TAEeluate.)

c) Klenow Repair

The precipitated DNA pellets were resuspended in a 50 ul Klenow reactionmixtur comprising:.

-   -   5 ul polynucleotide kinase buffer (10×),    -   0.5 ul 3.3 mM dNTPs (final concentration 33 uM),    -   at least 100 ng of purified PCR fragment,    -   distilled water to 50 ul, and    -   5 units Klenow DNA polymerase.        (10× means that the reagent was added at 10 times the desired        final concentration in the reaction mixture) Incubation was        carried out for 30 min at 37° C., followed by heat inactivation        for 10 min at 75° C.

The Klenow reaction repairs the ends where the BamHI restriction sitesare located. T4 DNA polymerase should not be used for this reaction asit will remove the sites by its exonuclease activity.

d) Kinasing of Ends

To the above reaction mixture, the following were added:

-   -   5 ul 100 mM rATP, and    -   10 units T4 polynucleotide kinase.        This reaction mixture was incubated at 37° C. for 30-60 min and        heat inactivated as before.        e) Concatemerization

The PCR products were then concatemerized to internalise the BamHIrestriction sites within large multimers. To the phosphorylationreaction, the following were added:

-   -   6 ul 10× ligase buffer, and    -   5 units of T4 DNA ligase.

The ligation was incubated overnight at 15° C. The concatemerizationreaction was heat inactivated as described previously.

f) Restriction Digestion

The concatemerized PCR products were digested into monomers using BamHIrestriction enzyme which simultaneously exposed the cohesive termini.The following were added to the heat inactivated ligation reactionmixture:

-   -   6 ul 10× B buffer (Boehringer), and    -   10-20 units BamHI.

The digestion mixture was incubated overnight at 37° C. Although theenzyme is not totally inactivated by heat, the reaction mixture was heattreated as before anyway.

At this point, the DNA was purified by a Magic prep column to removecleaved ends. The DNA was eluted from the Magic prep column in 10 ul inorder to be as concentrated as possible.

g) Ligation to Vector

100 ng of bacterial plasmid vector pUC18 was used in the ligation. Theplasmid vector DNA had been BamHI-cleaved and purified using “Geneclean”(Bio 101), but not dephosphorylated. (We found that thedephosphorylation reaction lowered the ligation efficiency of the vectorto a great extent.) It was planned that blue-white colour selection asdescribed hereafter would be sufficient to identify clones. The ligationreaction mixture contained the following:

110 ul of purified insert DNA produced as above

-   -   5 ul plasmid vector DNA,    -   1.5 ul 10× ligase buffer, and    -   1 unit of ligase.

The reaction mixture was incubated overnight at 15° C.

h) Transformation of E. coli

Bacterial transformations were carried out using the cell strain, XL-1Blue (Stratagene). Cells were made competent for transformation bystandard calcium chloride methods and stored quick-frozen in aglycerol/calcium chloride suspension in 200 ul aliquots. 3 ul of theligation reaction product were used to transform 100 ul of rapidlythawed competent XL-1 Blue cells (10 min on ice, 2 min at 42° C., 1 hourat 37° C. with shaking after addition of 1 ml of L broth). The cellswere plated in 200 ul aliquots onto L agar plates containing X-Gal (20ug/ml), IPTG (0.1 mM), Ap (50 ug/ml) and Tet (12.5 ug/ml). The presenceof the chemicals IPTG (isopropyl-β-D-thiogalactopyranoside) and X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) in the media producesa blue colour in colonies which contain plasmids such as the pUC serieswhich encode the lacZ enzymic peptide. The insertion of cloned DNA intothe polylinker region of these plasmids interrupts the lacZ peptidesequence, thereby destroying the ability of the plasmid to produce theenzyme. Bacterial colonies which contain recombinant plasmids will,therefore, be white.

i) Analysis of White Colonies

Blue-white selection identified cell colonies which containedrecombinant plasmids. These colonies were picked and DNA was preparedfrom them by mini-plasmid preparations. Digestion of the DNA with BamHIconfirmed that the plasmids contained cloned inserts. The plasmid DNAwas purified by glassmilk and sequenced using the USB Sequenase kit withM13 forward and reverse primers.

j) Discussion

Although this protocol contains a large number of steps, it is simple toexecute. Further work has found that the method has a high degree ofreproducibility. Theoretically, this cloning strategy should not work ifthe PCR product contains an internal BamHI site. However, the NS4sequence which was obtained for type 6 did contain such an internalsite; and there are no obvious reasons why foreshortened type 6sequences were not in fact the predominant product of the cloningexperiment.

Figure 5 shows the DNA and amino acid sequences for two regions or NS4in respect at HCV types 1 to 6 (for comparison) and types 4 to 6.

EXAMPLE 3 Synthesis of NS4 Peptides

The following peptides within the NS4 region of HCV types 1 to 6 weresynthesised. Types 4 to 6 are novel sequences according to the presentinvention, whereas types 1 to 3 are presented for comparison purposesand use in a complete HCV serotyping assay. HCV type 1[H₂N-KPAIIPDREVLYREFDEM]₈K₄K₂K-COOH (MDL031) (SEQ ID NO. 26) HCV type 1[H₂N-KPAVIPDREVLYREFDEM]₈K₄K₂K-COOH (MDL033) (SEQ ID NO. 24) HCV type 1[H₂N-RPAVIPDREVLYQEFDEM]₈K₄K₂K-COOH (MDL036) HCV type 1[H₂N-RPAVVPDREVLYQEFDEM]₈K₄K₂K-COOH (MDL035) HCV type 1[H₂N-ECSQHLPYIEQGMMLAEQF]₈K₄K₂K-COOH (MDL037Q) HCV type 1[H₂N-ECSQHLPYIEQGMALAEQF]₈K₄K₂K-COOH (MDL038Q) HCV type 2[H₂N-RVVVTPDKEILYEAFDEM]₈K₄K₂K-COOH (MDL039) HCV type 2[H₂N-ECASRAALIEEGQRIAEML]₈K₄K₂K-COOH (MDL041) (SEQ ID NO. 42) HCV type 2[H₂N-ECASKAALIEEGQRMAEML]₈K₄K₂K-COOH (MDL040) HCV type 3[H₂N-KPALVPDKEVLYQQYDEM]₈K₄K₂K-COOH (MDL042) (SEQ ID NO. 30) HCV type 3[H₂N-ECSQAAPYIEQAQVIAHQF]₈K₄K₂K-COOH (MDL044) (SEQ ID NO. 44) HCV type 4[H₂N-QPAVIPDREVLYQQFEDEM]₈K₄K₂K-COOH (MDL034) (SEQ ID NO. 32) HCV type 4[H₂N-ECSKHLPLVEHGLQLAEQF]₈K₄K₂K-COOH (MDL028) (SEQ ID NO. 46) HCV type 5[H₂N-RPAIIPDREVLYQQFDKM]₈K₄K₂K-COOH (MDL024) (SEQ ID NO. 34) HCV type 5[H₂N-ECSTSLPYMDEARAIAGQF]₈K₄K₂K-COOH (MDL029) (SEQ ID NO. 48) HCV type 6[H₂N-KPAVVPDREILYQQFDEM]₈K₄K₂K-COOH (MDL025) (SEQ ID NO. 36) HCV type 6[H₂N-ECSRHIPYLAEGQQIAEQF]₈K₄K₂K-COOH (MDL022) (SEQ ID NO. 50)(a) Synthesis of Multiple Antigenic Peptide MDL029.

In order to work successfully, the serotyping assay requires thatpeptides are synthesized on a special resin support, bearing themultiple antigen peptide core (K₄K₂K) as developed by Tam (Tam J. P.,1988, Proc.Natl. Acad. Sci.USA., 85:5409:5413). All peptides weresynthesized on an Applied Biosystems model 432A Synergy peptidesynthesizer running FASTmoc™ chemistry. Peptide synthesis was achievedusing the standard run program without modification. All reagents usedwere supplied by Applied Biosystems Limited (Kelvin Close, BirchwoodScience Park, Warrington, UK). The MAP resin was hepta-lysyl (K₄K₂K)core on a polyoxyethylene/polystyrene co-polymer with an HMP linker andβ-alanine internal reference amino acid. The N-a-amino group of allamino acids was protected by the 9-fluorenylmethoxycarbonyl (Fmoc)group. Amino acids with reactive side groups were protected as follows;Amino Acid Code Protecting Group glutamine Q tert. Butyl ester (OtBu)cysteine C trityl (Trt) serine S tert. Butyl (tBu) threonine T tert.Butyl (tBu) tyrosine Y tert. Butyl (tBu) aspartic acid D tert. Butylester (OtBu) arginin R 2,2,5,7,8-Pentamethylchroman- 6-sulphonyl (Pmc)

The progress of the synthesis was monitored by measuring theconductivity of the coupling and deprotection mixtures. There are noabnormalities in the conductivity trace and the synthesis was consideredsuccessful.

Following the synthesis, the fully protected peptide resin wastransferred to a conical 50 mL capacity polypropylene tube and treatedwith thioanisole (0.15 mL), ethandithiol (0.15 mL) and trifluoroaceticacid (TFA; 2.7 mL). The mixture was stirred for three hours at roomtemperature, then filtered through glass wool in a Pasteur pipette, thefiltrate dropping into 20 mL tert. butylmethylether (TMBE) contained ina glass screw-capped bottle, whereupon the peptide precipitated. Thetube was centrifuged and the liquor aspirated, leaving the peptidepellet in the tube. Peptide was washed with three further 20 mL aliquotsof TMBE, using a spatula to dis-aggregate the pellet and centrifugationto recover the peptide. The peptide was finally dried at roomtemperature in vacuo.

To analyse the purity of the peptide, a small sample was dissolved inpurified water and submitted to analysis by reverse phasehigh-performance liquid chromatography (HPLC). The reverse phase columnwas 250×4.6 mm containing a C₄ matrix. Peptide was eluted with agradient of 3.5% acetonitrile to 70% acetonitrile over 20 minutes at aflow rate of 1.5 mL min⁻¹. The eluant was monitored at 214 nm to detectpeptide.

EXAMPLE 4 Assay for the Determination of HCV Serotypes 1-6

We have developed an assay based on selective competition, which iscapable of distinguishing the serotype of HCV to which antibodiespresent in a biological sample have been produced. Typically the samplewill be serum or plasma from a human with confirmed hepatitis Cinfection.

The assay relies on the selectivity of 17 different synthetic peptidescovering variable sequences within the NS4 protein of hepatitis C virustypes 1,2,3,4,5 & 6. Whilst there is a degree of cross-reactivitybetween antisera raised to NS4 of an HCV serotype and the homologousregion of other serotypes, this can be blocked without completelyremoving the true reactivity. In view of this, we have developed anassay format involving coating wells of a microtitre plate with an equalamount of all 17 peptides. Samples are tested in eight duplicate wellswhich each receive a different mixture of blocking peptides. Only onetesting well and the un-completed control well should containcolouration at the end of the assay protocol, thereby identifying theserotype of the infecting hepatitis C virus.

Plates for serotyping assays are prepared by coating approximatelyequimolar amounts of each of the peptides onto polystyrene microwellplates. Peptides are dissolved in purified water and a mixture is madeat the following concentration: MDL031 25 ng/ml MDL033 MDL036 {closeoversize parenthesis} MDL035 MDL037Q MDL038Q MDL039 50 ng/ml MDL041MDL040 MDL042 MDL044 MDL034 {close oversize parenthesis} MDL028 MDL024MDL029 MDL025 MDL022Although strictly speaking each microwell need only contain peptide ofthe type which it is intended to detect, it is more convenient to coateach microwell with all six antigen types.

Peptides were allowed to bind to the plates by adding 100 ul of thepeptide mixture into each well of the plate and incubating at +4° C.overnight.

To provide sufficient differentiation between the various serotypes, itis then necessary to add competing heterologous peptides to the samplebeing tested. Competing solutions are made up in assay sample diluent togive a 100-fold excess of competing peptide, relative to the coatingconcentration. The different competing solutions are classifiedaccording to the serotyp for which they are blocking, i.e. CompetingSolution 1 contains peptides for types 2-5. Competing solutions are madeto have the excess required in 10 ul. The competing solutions are asfollows: Competing solution Peptide Concentration 1 MDL039 50 ug/mlMDL041 ″ MDL040 ″ MDL042 ″ MDL044 ″ MDL034 ″ MDL028 ″ MDL024 ″ MDL029 ″MDL025 ″ MDSL022 ″ 2 MDL031 25 ug/ml MDL033 ″ MDL036 ″ MDL035 ″ MDL037Q″ MDL038Q ″ MDL042 50 ug/ml MDL044 ″ MDL034 ″ MDL028 ″ MDL024 ″ MDL029 ″MDL025 ″ MDL022 ″ 3 MDL031 25 ug/ml MDL033 ″ MDL036 ″ MDL035 ″ MDL037Q ″MDL038Q ″ MDL039 50 ug/ml MDL040 ″ MDL041 ″ MDL034 ″ MDL028 ″ MDL024 ″MDL029 ″ MDL025 ″ MDL022 ″ 4 MDL031 25 ug/ml MDL033 ″ MDL036 ″ MDL035 ″MDL037Q ″ MDL038Q ″ MDL039 50 ug/ml MDL040 ″ MDL041 ″ MDL042 ″ MDL044 ″MDL024 ″ MDL029 ″ MDL025 ″ MDL022 ″ 5 MDL031 25 ug/ml MDL033 ″ MDL036 ″MDL035 ″ MDLo37Q ″ MDL038Q ″ MDL039 50 ug/ml MDL040 ″ MDL041 ″ MDL034 ″MDL028 ″ MDL042 ″ MDL044 ″ MDL025 ″ MDL022 ″ 6 MDL031 25 ug/ml MDL033 ″MDL036 ″ MDL035 ″ MDL037Q ″ MDL038Q ″ MDL039 50 ug/ml MDL040 ″ MDL041 ″MDL034 ″ MDL028 ″ MDL024 ″ MDL029 ″ MDL042 ″ MDL044 ″

-   -   1) Add 180 ul sample of diluent to each well.    -   2) Add 10 ul blocking peptides to relevant wells.    -   3) Add 10 ul sample to each of six wells.    -   4) Mix plate and incubate at 37° C. for 1 hour.    -   5) Wash wells three times.    -   6) Add 100 ul conjugate to each well.    -   7) Incubate at 37° C. for 1 hour.    -   8) Wash wells three times.    -   9) Add 100 ul TMB solution (including        3,31,5,5′-tetramethylbenzidine, hydrogen peroxide, buffers etc).    -   10) Incubate at 37° C. for 30 minutes.    -   11) Stop reaction with sulphuric acid; and    -   12) Read optical density at 450 nm/690 nm.

Samples known to contain anti-HCV antibodies are tested at a dilution of1/20 with 100 fold excess of competing peptides, in a total volume of200 ul as described above. Following incubation, sample is removed andthe wells are washed. An anti-human immunoglobulin G conjugated tohorseradish peroxidase is added, and binds to any captured anti-HCVantibodies. Bound antibody is then visualised by removing the enzymeconjugate and adding a substrate and chromogen, which, in the presenceof enzyme, converts from a colourless to a coloured solution. Theintensity of the colour can be measured and is directly proportional tothe amount ot enzyme present. Results on certain samples are given inTable

EXAMPLE 5 Assay for the Determination of HCV Serotypes 4-6

An alternative to the assay format in Example 4 has been developed. Thisassay is more limited in scope using only the peptides for types 4,5 &6. For some samples, the more comprehensive assay may give erroneousresults, due to greater cross-reactivity with type 1 peptides. Theprotocol for performing the more restricted assay is identical to thatgiven in Example 4.

Plates for the HCV types 4-6 assay are prepared as described in Example4 with the only difference being the reduced number of peptides used.Peptides are dissolved in purified water and a mixture is made at thefollowing concentration: MDL034 50 ng/ml MDL028 MDL024 {close oversizeparenthesis} MDL029 MDL025 MDL022Peptides are allowed to bind to the plates by adding 100 ul of thepeptide mixture into each well of the plate and incubating at +4° C.overnight.

To provide sufficient differentiation between the various serotypes, itis then necessary to add competing heterologous peptides with the samplebeing tested. Competing solutions are made up in assay sample diluent togive a 100-fold excess of competing peptide, relative to the coatingconcentration.

The different competing solutions are classified according to theserotype for which they are blocking, i.e. Competing Solution 4 containspeptides for types 5 & 6. Competing solutions are made to have theexcess required in 10 ul. The competing solutions are as follows:Competing Solution Peptide Concentration 4 MDL024 50 ug/ml MDL029 ″MDL025 ″ MDL022 ″ 5 MDL034 50 ug/ml MDL028 ″ MDL025 ″ MDL022 ″ 6 MDL03450 ug/ml MDL028 ″ MDL024 ″ MDL029 ″

Samples known to contain anti-HCV antibodies are tested at a dilution of1/20 with 100 fold excess of competing peptides, in a total volume of200 ul, as described in Example 4. Following incubation, sample isremoved and the wells are washed. An anti-human immunoglobulin Gconjugated to horseradish peroxidase is added, and binds to any capturedanti-HCV antibodies. Bound antibody is then visualised by removing theenzyme conjugate and adding a substrate and chromogen, which, in thepresence of enzyme, converts from a colourless to a coloured solution.The intensity of the colour can be measured and is directly proportionalto the amount of enzyme present.

Results on certain samples are given in Table 2. TABLE 1 NO ALL TYPETYPE TYPE TYPE TYPE TYPE SAMPLE BLOCK BLOCK 1 2 3 4 5 6 RESULT Control 10.515 0.049 0.530 0.033 0.038 0.038 0.031 0.029 1 Control 2 1.821 0.0100.028 1.916 0.004 −0.007 0.040 0.005 2 Control 3 2.035 0.066 0.048 0.0461.933 0.048 0.067 0.044 3 Egypt 3 OVER 0.372 0.346 0.478 0.322 OVER0.256 0.430 4 C1016116 OVER 0.456 0.635 0.480 0.533 0.533 OVER 0.617 5Control 6 0.082 0.004 0.007 −0.003 −0.004 −0.012 −0.011 0.067 6 PD372OVER 0.076 1.497 0.081 0.072 0.040 0.057 0.075 1 PD513 OVER 0.548 0.467OVER 0.553 0.470 0.525 0.582 2 AD558 OVER 0.031 0.064 0.044 OVER 0.0350.054 0.048 3 Egypt 37 OVER 0.034 0.064 0.039 0.045 1.473 0.047 0.037 4C1015921 0.173 0.010 0.017 0.008 0.011 0.005 0.182 0.012 5Positive results are shown in bold text.

TABLE 2 NO ALL TYPE TYPE TYPE SAMPLE BLOCK BLOCK 4 5 6 EGYPT 1 OVER0.109 1.669 0.184 0.048 EGYPT 2 0.525 0.010 0.514 0.001 −0.006 EGYPT 3OVER 0.269 OVER 0.329 0.201 EGYPT 4 0.266 −0.001 0.292 0.006 −0.002EGYPT 5 1.677 0.090 1.274 0.094 0.086 EGYPT 6 OVER 0.208 0.317 0.0410.022 EGYPT 7 OVER 0.113 OVER 0.143 0.204 EGYPT 8 OVER 0.085 0.891 0.1050.098 EGYPT 9 0.051 0.009 0.055 0.014 0.022 EGYPT 10 OVER 0.021 2.0230.043 0.073 EGYPT 11 0.077 0.024 0.095 0.032 0.048 C1016116 OVER 0.6470.778 OVER 0.513 C1015921 0.196 0.014 0.009 0.222 0.009 C1132558 0.0150.007 0.008 0.015 0.007 K904836 OVER 0.073 0.101 OVER 0.071 Control 60.433 0.029 0.013 0.015 0.314 HK T3950 0.428 0.034 0.017 0.027 0.366 HKT3943 OVER 0.348 0.424 0.342 1.105Positive results are shown in bold text.NB - The preponderence of Type 4 results reflects the relative incidenceand availability or samples for types 4, 5, & 6.

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1-41. (Canceled)
 42. An isolated peptide having an antigenic sequence selected from the following: a) QPAVIPDREVLYQQFDEM; (SEQ ID NO: 32)

and minor variations thereof.
 43. A peptide according to claim 42 which is bound to a multiple antigen peptide core.
 44. A peptide according to claim 43 having a sequence selected from the following: a) [H₂N-QPAVIPDREVLYQQFDEN]₈K₄K₂K- (SEQ ID NO: 32) COOH; and b) [H₂N-ECSKHLPLVEHGLQLAEQF]₈K₄K₂K- (SEQ ID NO: 46) COOH;

where K₁K₂K is the multiple antigen peptide core.
 45. A peptide according to claim 42 which is fused to another peptide to form a fusion peptide.
 46. A peptide according to claim 45 fused to another peptide selected from the group consisting or β-calactosidase, glutathione-S-transferase, trpE and polyhedrin coding sequence.
 47. A peptide according to claim 42, wherein said peptide is labelled.
 48. (Canceled)
 49. A vaccine formulation which comprises a peptide according to claim
 42. 50. An immunoassay device which comprises a solid substrate having immobilized thereon a peptide according to claim
 42. 51. A device according to claim 50 wherein a mixture of antigenic peptides of HCV type 4, type 5 or type 6 is immobilized on the solid substrate.
 52. A device according to claim 50 wherein a mixture of antigenic peptides of HCV types 4, 5 and 6 is immobilized on the solid substrate.
 53. A device according to claim 50 wherein the mixture further comprises one or more antigenic NS4 peptides of HCV types 1 to
 3. 54. A device according to claim 53 wherein the mixture is a mixture of HCV type 1, 2, 3, 4, 5, and 6 antigenic peptides.
 55. A device according to claim 50 for HCV typing which comprises a solid substrate containing HCV-4, HCV-5 and HCV-6 antigenic peptides.
 56. (Canceled)
 57. A device according to claim 50 is further comprising a mixture of non-immobilized heterologous-type blocking HCV peptides.
 58. A device according to claim 54 for the detection or HCV types 1 to
 6. 59. An immunoassay kit which comprises an immunoassay device according to claim 57, together with a series of solutions, each solution comprising a mixture of heterologous-type blocking HCV peptide.
 60. An immunoassay kit as claimed in claim 59, wherein the immunoassay device comprises a solid substrate having having immobilized thereon a mixture of antigenic peptides of HCV types 1,2,3,4,5, and 6; together with a series of six competing solutions, each solution containing a mixture or different antigenic peptides of HCV types 1,2,3,4,5, and
 6. 61. (Canceled)
 62. A method of in vitro screening a sample for HCV antibodies which comprises: a) obtaining said sample: b) contacting said sample with a peptide of claim 22, and c) detecting any antibody-antigen complex produced.
 63. A method according to claim 62 wherein the peptide is immobilized on a solid substrate.
 64. A method according to claim 63 wherein a mixture of peptides is immobilized on the solid substrate.
 65. A method according to claim 64 wherein the mixture is a mixture of HCV type 1,2,3,4,5 and 6 antigenic peptides.
 66. A method according to claim 63 wherein the sample and a mixture of heterologous-type blocking HCV peptides are applied to the peptide immobilized on the solid substrate. 67-68 (Canceled)
 69. A method according to claim 62, wherein HCV antibodies present in the sample are captured on a solid substrate, wherein said peptide is labeled, and wherein said peptide is applied to said HCV antibodies captured on said substrate for detection of any captured HCV antibodies. 70-72 (Canceled) 